This work focuses on electrophoretic variants of arylsulfatase A (ASA) which have been proposed as a predisposing factor for dysmyelination due to alcohol intake. The studies in this grant application will examine structural, kinetic, and biological consequences of normal and variant ASA's. Extensive structural studies of the glycan and primary polypeptide structure will be performed. The effect of specific hydrolytic enzymes, lectin binding, and mannose-6-phosphate receptor binding on ASA will be studied to explore the structural elements of the glycan moiety. Since previous work has delineated the importance of the phosphorylated oligomannose glycan of the normal ASA on electrophoretic migration, particular attention will be paid to this parameter for the variant ASA's. The purified proteins will be fragmented and subjected to high performance liquid chromatography. Individual peaks which differ between the variant and normal enzymes will be analyzed in terms of both amino acid and carbohydrate content and sequence. The kinetic parameters of the variant and normal ASA's will be assessed in vitro using sulfatide as substrate in the presence of activator protein. A galactose biosensor will be used to determine the concentration of cerebroside, the product of the ASA reaction. The effect of ethanol and acetaldehyde on the reaction will also be measured. The biological consequences of ethanol on sulfatide metabolism and subcellular localization of normal and variant enzyme will be determined within fibroblast cells grown from skin biopsies. Subcellular localization of ASA will be performed by electron microscope cytohistochemistry as well as immunofluorescence analysis utilizing specific anti-ASA serum. In addition, high resolution subcellular fractionation will be performed to identify biochemically the location of ASA. In vitro cultured fibroblasts will be employed to metabolically label ASA with radioactive sulfate, phosphate, or glucosamine in order to assist in identifying sulfation and phosphorylation patterns of the normal and variant enzyme, as well as to allow detection of small amounts of glycan for complete structural analysis of ASA variants. It is anticipated that the integrated results of these various approaches will demonstrate structural and functional differences between the normal and variant ASA's.